PROJECT SUMMARY: The loss of lung endothelial cell (EC) barrier integrity, resulting in vascular leakage and alveolar flooding, is a critical feature of the pathobiology of ventilator-induced lung injury (VILI). Studies by the Project #3 team were the first to demonstrate that sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, is an effective therapy for reducing vascular leakage in inflammatory lung injury via ligation of S1P1, a G-protein-coupled S1P receptor highly expressed in lung ECs. We have demonstrated that ligation of the S1P1 receptor by S1P or S1P analogues, rapidly stimulates a signaling cascade that reorganizes the lung EC cytoskeleton via Rac1 GTPase activation, enhances junctional integrity and decreases vascular permeability. S1P administration re- duces murine and canine lung vascular hyper-permeability evoked by VILI, LPS, ischemia/reperfusion, radia- tion, or traumatic brain death with marked improvement in oxygenation and alveolar edema formation. In con- trast, ligation of the S1P3 receptor induces Rho GTPase signaling to the cytoskeleton to increase lung perme- ability. Furthermore, S1P3 is released into circulating microvesicles by VILI or LPS and serves as a novel ARDS biomarker that predicts survival. Our published and preliminary data indicate that physiologic and path- ophysiologic levels of mechanical stress have selective effects on S1PR1 and S1PR3 promoter activity, ex- pression and downstream signaling. This includes selective promoter demethylation responses to excessive mechanical stress and the influence of promoter single nucleotide polymorphisms (SNPs) on promoter activity. In Project #3, we will facilitate the translation of information on the role of S1P1/S1P3 in EC responses to me- chanical stress and VILI, and evaluate the role of S1P1 and S1P3, downstream effectors (Rac1 and RhoA), the influence of key S1P1 and S1P3 post-translational modifications (PTMs), and the contribution of S1PR1 and S1PR3 SNPs to VILI risk and severity. Specific Aim (SA) #1 will detail S1PR1 and S1PR3 promoter regulatory elements, sites of promoter methylation induced by mechanical stress and the influence of S1PR1 and S1PR3 promoter SNPs on mechanical stress-induced S1PR1 & S1PR3 5' promoter regulation and expression. Our published studies have also indicated that VILI is a major stimulus for ROS generation and protein nitration with S1P3 a specific target. SA #2 will characterize the functional consequences of mechanical stress-induced PTMs (nitration and phosphorylation) involved in S1P1/S1P3 dysregulated Rac1 and RhoA signaling cascades that alter lung vascular barrier integrity. These studies will include effects of S1PR1 and S1PR3 coding SNPs, evaluation of S1P3 nitration, persistently activated nitrated RhoA, and persistently inactivated nitrated Rac1, as novel and effective VILI biomarkers and signaling molecules. Finally, SA #3 will evaluate potential VILI thera- peutic targets including novel S1P1-specific agonists, and Rac1 and RhoA nitration-shielding peptides. To- gether, Project #3 studies will validate novel therapies targeting this complex signaling pathway in the resolu- tion of VILI pathobiology and advance strategies to reduce the morbidity and mortality of VILI/ARDS.